In human cells, all membrane proteins (which include many if not most drug targets) and all peptide and protein hormones (including insulin and leptin) travel through the endomembrane system from the place where they are made in the cell to the place where they do their work. Diseases thought to involve the endomembrane system include Alzheimer's dementia, cystic fibrosis, and several diseases of fat and cholesterol metabolism. The endomembrane system itself consists of 3 major parts: the endoplasmic reticulum (ER), the Golgi apparatus and the plasma membrane, plus a complex series of internalization and recycling pathways grouped under the general heading of endocytosis. The long term goal of this research is to build a computer model of the secretory pathway and the endocytosis system that has been successfully tested against experimental results, and can be used to make new predictions. Our short-term goal is to test the possible roles of cholesterol in controlling the endomembrane system. By combining the live-cell imaging of fluorescent proteins and cholesterol with systems biology and kinetic modeling we will test the hypothesis that cholesterol-driven membrane partitioning is the organizing principle of the endomembrane system. The focus will be on testing this idea in four specific areas: 1) cholesterol-regulation of protein export from the endoplasmic reticulum, 2) determining whether the Golgi apparatus or lipid transfer proteins carry cholesterol from the ER to the plasma membrane, 3) cholesterol driven partitioning in the Golgi apparatus, and 4) cholesterol trafficking pathways in endocytosis. Because the goal is an integrated model of these important cell processes, the model will also be tested against key experimental results from other laboratories world-wide. PUBLIC HEALTH RELEVANCE: Proteins are responsible for making the human body work. More than half of our proteins could not get to the place where they do their work without the "secretory pathway." Diseases of the secretory pathway include Alzheimer's dementia, cystic fibrosis and several diseases of fat metabolism. The goal of our project is to discover how the secretory pathway works by using computer modeling to combine results from many scientists.